Background Menopause is associated with an increase in the prevalence and severity of hypertension in women. Although premenopausal females are protected against T cell‐dependent immune activation and development of angiotensin II (Ang II) hypertension, this protection is lost in postmenopausal females. Therefore, the current study hypothesized that specific CD4 + T cell pathways are regulated by sex hormones and Ang II to mediate progression from premenopausal protection to postmenopausal hypertension. Methods and Results Menopause was induced in C57BL/6 mice via repeated 4‐vinylcyclohexene diepoxide injections, while premenopausal females received sesame oil vehicle. A subset of premenopausal mice and all menopausal mice were infused with Ang II for 14 days (Control, Ang II, Meno/Ang II). Proteomic and phosphoproteomic profiles of CD4 + T cells isolated from spleens were examined. Ang II markedly increased CD4 + T cell protein abundance and phosphorylation associated with DNA and histone methylation in both premenopausal and postmenopausal females. Compared with premenopausal T cells, Ang II infusion in menopausal mice increased T cell phosphorylation of MP2K2, an upstream regulator of ERK, and was associated with upregulated phosphorylation at ERK targeted sites. Additionally, Ang II infusion in menopausal mice decreased T cell phosphorylation of TLN1, a key regulator of IL‐2Rα and FOXP3 expression. Conclusions These findings identify novel, distinct T cell pathways that influence T cell‐mediated inflammation during postmenopausal hypertension.
T cells are involved in hypertension pathogenesis in both males and postmenopausal females while premenopausal females are resistant to T cell-mediated Ang II-induced hypertension. The goals of this study were (1) to identify T cell specific proteomic pathways associated with postmenopausal susceptibility to hypertension (2) to identify T cell specific transcriptomic changes associated with premenopausal protection from hypertension. Proteomic analysis was performed on splenic CD4 + T cells isolated from premenopausal and postmenopausal females (VCD, 160 mg/kg/day i.p. 20d) following Ang II infusion (800 ng/kg/min 14d). 384 proteins from CD4 + T cells were identified as differentially expressed following Ang II infusion in premenopausal females. 285 proteins from CD4 + T cells were identified as differentially expressed between premenopausal and postmenopausal females following Ang II infusion. Gene ontology (GO) analysis of pre vs. postmenopausal proteins identified enriched pathways associated with RNA binding, chaperone activity and cellular stress responses. Transcriptomic changes were analyzed, via RNAseq, on isolated splenic CD4 + T cells from premenopausal females, with and without Ang II infusion. Thirty-four genes were identified as differentially expressed in CD4 + T cells following Ang II infusion. GO analysis of Ang II upregulated genes revealed an enrichment of five distinct molecular functions, including antioxidant activity. In a subsequent study to validate the RNAseq, we confirmed that Ang II increased CD4 + T cell mRNA expression of calprotectin (S100a8/S100a9), a calcium and zinc binding protein complex that contributes to antioxidant defense (S100a8: Con 1.0 ± 0.4 vs Ang II 5.0 ± 0.8*; S100a9: Con 1.0 ± 0.4 vs Ang II 6.0 ± 0.8*, *P<0.05 vs Con). Furthermore, we determined that Ang II did not increase calprotectin expression in mice lacking estrogen receptor α (ERKO) (S100a8: ERKO 0.1 ± 0.4 vs ERKO/Ang II 0.3 ± 0.7; S100a9: ERKO 0.2 ± 0.5 vs ERKO/Ang II 0.3 ± 0.6). The current studies demonstrate a role for estrogen in Ang II-induced T cell gene expression and signal transduction, and begin to elucidate the molecular mechanisms of female protection from T cell-mediated hypertension.
Cycling female mice are protected against Angiotensin II (Ang II) hypertension, and inducing ovarian failure (menopause) eliminates this protection. T lymphocytes are required for the development of Ang II hypertension in male mice, however premenopausal females are resistant to T cell‐induced hypertension. Following menopause (loss of estrogen) resistance is lost, allowing activation of T cell‐induced hypertension in postmenopausal females. The purpose of this study was to identify, in vivo, T cell‐specific proteome responses to Ang II, before and after menopause. 10‐week‐old C57BL/6 female mice received i.p. 4‐vinylcyclohexene diepoxide (VCD) injections for 20 consecutive days to induce ovarian failure (VCD menopause model). Cyclicity was monitored daily via vaginal cytology. Once in menopause, Ang II was infused (800 ng/kg/min) for 14 days, which has been shown to cause an increase in SBP of 25 mmHg. Ang II in VCD‐treated menopausal females (VCD/AngII) resulted in significantly decreased heart rates versus controls (Control 682 ± 5.6 vs. VCD/AngII 592.5 ± 28.5, p < 0.05). Splenic CD4+ T cells were purified via negative immune‐magnetic selection and CD4+ purity was measured via flow cytometry (>90% purity). Proteomic analysis was performed on control, pre‐menopausal/Ang II and VCD/AngII mice (n=4 per group). Protein lysates from the CD4+ T cells were separated by SDS‐PAGE and subjected to in‐gel tryptic digestion followed by tandem mass spectrometry analysis, resulting in 7,123 proteins identified across the entire experiment. Quantitative proteomics were performed via label‐free quantification using Progenesis software‐based extracted ion abundance. Of the 5,857 proteins identified with two or more unique peptides, 474 of the proteins exhibited significant abundance differences between control, Ang II and VCD/Ang II groups as assessed by One‐Way ANOVA statistical analysis (p ≤ 0.05). Gene Ontology (GO) enrichment analysis of these 474 proteins identified 159 GO biological pathways that were significantly overrepresented (p ≤ 0.05). Pathways associated with increased inflammation and reduced inhibition of inflammatory cytokines were among those identified. Using IHC we show that renal macrophage infiltration is significantly increased in VCD/Ang II mice compared to Ang II and control groups. Overall, our data suggests that following Ang II infusion, splenic T cells are differently impacted by the loss of estrogen, leading to an increased pro‐hypertensive cytokine milieu compared to pre‐menopausal Ang II infused mice.Support or Funding InformationR01HL131834 (HLB)Sarver Heart Center Endowment for Women's Health Research (HLB)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of T cell-dependent pro-inflammatory responses are required for Ang II hypertension in male mice. However, females are protected from T cell-mediated hypertension and may suppress hypertension by directly preventing Ang II-induced pro-inflammatory T cell activation. Here we sought to determine whether transferring T cells from hypertensive donor mice eliminates female protection against T cell-mediated hypertension. Splenic CD3 + T cells were transferred from normotensive (NT) or Ang II-hypertensive (HT) C57BL/6J male donors to female Rag-1 -/- (NT T cell female-NTF; HT T cell female-HTF) or male Rag-1 -/- (HT T cell male-HTM) recipient mice. Blood pressure was monitored (tail cuff) for 5 weeks post-transfer. Ang II (490ng/kg/min) was infused into recipient mice for 14 days during weeks 4 and 5 post-transfer (NTFA; HTFA; HTMA). Ang II significantly increased MAP in donor male mice (NT 114 vs HT 157 mmHg, p<0.05). Transfer of T cells from HT donors did not induce HT in female or male recipients. Similarly, T cell donor environment did not affect Ang II-induced blood pressure in female recipients, which remained protected compared to male recipients (MAP: NTF 83 + 4 mmHg*, HTF 88 + 6 mmHg*, NTFA 101 + 5 mmHg*, HTFA 103 + 5 mmHg*, HTMA 138 + 3 mmHg, *p<0.05 vs HTMA). Flow cytometry demonstrated similar splenic T cell frequency across all groups (CD3: NTF 18%, NTFA 16%, HTF 17%, HTFA 14%, HTMA 18%, p>0.05). However, regulatory T cells were significantly reduced in male recipients compared to all female groups (Foxp3: NTF 21.6%*, NTFA 22.2%*, HTF 22.8%*, HTFA 22.6%*, HTMA 15.3%, *p<0.05 vs HTMA) Females had significantly less renal T cell infiltration compared to males and infiltration was not impacted by Ang II infusion or T cell donor status (CD3: NTF 12,083*, NTFA 11,317*, HTF 12,656*, HTFA 8,997*, HTMA 22,405, *p<0.05 vs HTMA; CD4: NTF 6,411*, NTFA 4,702*, HTF 5,831*, HTFA 4,579*, HTMA 9,914, *p<0.05 vs HTMA; CD8: NTF 5,397*, NTFA 6,123*, HTF 6,362*, HTFA 3,792*, HTMA 11,727, *p<0.05 vs HTMA). These results demonstrate that female mice prevent T cell-mediated hypertension and renal T cell infiltration regardless of previous T cell exposure to a hypertensive environment, suggesting a direct preventive mechanism in females against pro-hypertensive T cell responses.
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